HIV-1 Vif in AIDS-Related Malignancies ABSTRACT Viral infectivity factor (Vif) is an accessory protein found in nearly all lentiviruses including HV-1 and is essential for productive infection in CD4+ T cells. Its canonical function revolves around the APOBEC3-Vif axis, whereby Vif polyubiquitinates and targets APOBEC3 proteins for degradation by hijacking the transcription factor CBF and an E3 ubiquitin ligase containing CUL5, ELOB, ELOC, and RBX2. However, numerous data suggest that Vif possesses alternative functions. Recently, we have found that Vif also interacts with the autophagy-related protein, AMBRA1, and that this Vif-AMBRA1 interaction is conserved among HIV-1, SIV, FIV, BIV, and MVV. Conservation alone suggests a novel alternative Vif function. Vif most likely hijacks AMBRA1 from its normal functions in apoptosis and autophagy and forms an E3 ubiquitin ligase to degrade cellular proteins. Aim 1 of this proposal will focus on identifying the complete Vif-AMBRA1 interacting complex by 1A) using a validated tandem affinity purification-mass spectrometry approach and 1B) mapping the interaction domains of each constituent via systematic truncation and site-directed mutagenesis. Aim 2 of this proposal will explore the function of the Vif-AMBRA1 complex by 2A) quantifying the impact of Vif on AMBRA1- mediated autophagy and apoptosis and 2B) identifying the cellular ubiquitination targets of the Vif-AMBRA1 ligase complex. The studies proposed here may provide an additional mechanism of tumorigenesis during HIV infection. As is with all tumors, no one-step process directly explains AIDS-related malignancies, but combinations of factors such as subversion of host proteins and modulation of cellular processes serve as important steps in this complex process.